In vitro Culture of Heat-treated Anthers Induces Embryogenic Callus in Cassava (Manihot esculenta Crantz)

Abstract

Microspore embryogenesis is the developmental plasticity of juvenile male gametophytes to switch from pollen to embryo development upon exposure to in vitro stress. It is a common method for obtaining haploid and doubled haploid plants in breeding programs for development of superior varieties via haploid-diploidization which allows fast development of homozygous lines from heterozygous parents. In cassava, obtaining haploidization through traditional methods of successive selfing is difficult because of cassava’s long reproductive cycle, high heterozygosity, and inbreeding depression. As a first step towards microspore embryogenesis, callus induction following heat treatment has not been investigated in cassava. We used two elite Ugandan cassava varieties, hereafter called “genotypes”, NASE3 and NASE14, for the study of callus induction. Heat stress of 40°C for 0, 6, 12, 18 and 24hrs and Murashige and Skoog medium supplemented with 2-9% sucrose, and 2,4-dichlorophenoxy acetic acid were used. Heating anthers of NASE3 at 40°C for 6 hrs resulted in a significantly higher percentage of callus induction on MS medium supplemented with 2% sucrose. Callus emerged from inside of the anthers with production influenced by genotype, sucrose concentration, anther density and duration in culture (P≤0.001). Limited in vitro callus differentiation was observed on auxin-and cytokinin-supplemented media. In both genotypes, embryo genic callus was obtained in liquid medium, while green callus was achieved on solid medium. This is a significant step upstream of double haploid plant production pathway in cassava: rigorous optimization of protocols downstream of callus induction are needed for regeneration of microspore-derived embryos and haploid plants.